Apparatus and method for culturing and preserving tissue constructs

ABSTRACT

A disclosure is made of various apparatus and methods for culturing and preserving cells and tissue in ways that minimize contamination potential, direct cells to reside in desired areas, allow uniform cell distribution during seeding, provide optimal growth conditions by controlling the amount of medium residing in proximity of cells, allow desired compounds and molecules to reside in proximity of the cells, allow co-culture, provide for efficient scale up, allow a desired shape of tissue to be created while retaining a closed system, and allow cryopreservation and reconstitution of cell and tissue while retaining a closed system. The apparatus and methods can be combined to prevent the need to remove the tissue from the enclosure at any point during the sterilization, seeding, culturing, cryopreservation, shipping, or restoration process. Also disclosed is an apparatus and method of pipette interface with a container in a manner that blocks contaminants from entering the container.

RELATED APPLICATION

This application is a division of application Ser. No. 11/709,933 filedFeb. 22, 2007 which claims the benefit of application Ser. No.10/460,850 filed Jun. 13, 2003, now U.S. Pat. No. 7,229,820, whichclaims the benefit of U.S. Provisional Application No. 60/388,567 filedJun. 13, 2002.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to the culture, handling, preservation,storing, reconstitution, and shipping of cells and tissue. Specifically,the present invention relates to apparatus and methods for culturing andpreserving cells and tissue in an enclosure without the need to removethem from the enclosure at any point during the sterilization, seeding,culturing, cryopreservation, shipping, or restoration process. Alsodisclosed is an apparatus and method of pipette interface with acontainer in a manner that blocks contaminants from entering thecontainer.

2. Discussion of the Related Art

Tissue constructs hold promise to provide societal health care benefits.A wide variety of potentially beneficial applications are emerging, somewith the potential to repair defects or abnormal tissues in the body,including those related to wound care, cardiovascular disease, andorthopedic care. These applications are anticipated to result in atremendous amount of tissue constructs being produced annually to meetthe needs of society. The tissue culture device used for tissueconstruct culture plays a critical role in the cost of production, whichin turn impacts overall health care costs to society.

The petri dish has commonly been used to culture tissue constructs forresearch and production applications because of its simplicity.Unfortunately, the petri dish does not provide optimal cultureconditions or allow efficient production. The petri dish is subject tocontamination, automated handling is difficult, tissue constructsresiding in a petri dish are prone to gradient exposure, tissueconstructs must be physically handled during the manufacturing process,controlling the final shape of the tissue construct is difficult, andthe petri dish is not suited to efficient process control and qualitycontrol.

Another problem of the petri dish is related to inoculation of cellattachment matrices that facilitate three-dimensional culture such ascollagen. Unless the tissue construct is to be the same size and shapeas the petri dish, cells generally must be placed directly onto the cellattachment matrix, and not deposited gravitationally as is the case withmost cell culture devices, in order to prevent cells from coming toreside on the petri dish itself. Cells residing on surfaces other thanthe cell attachment matrix can negatively impact the culture. Thus, theinoculation procedure does not have tight process control since cellsmust be directed, usually manually, to specific locations on the cellattachment matrix. This problem is magnified as production is scaled upto produce more and more tissue constructs.

An example of a typical tissue construct culture process in a petridish, the culture of skin, illustrates the problems. One method ofculturing living skin in described by Eisenburg in U.S. Pat. No.5,282,859. A cross-linked collagen sponge is created in a manner thatrenders it porous on one side and having a non-porous skin on the otherside. Fibroblast cells are inoculated by “injecting” them onto theporous side of the collagen sponge, which resides in a petri dish.Culture medium is placed in the petri dish, which is incubated for 10days while the fibroblasts proliferate. The culture medium is replacedevery second day, requiring the device to be exposed to potentialcontamination on 5 separate occasions. It may be helpful to conditionthe medium by exposing it to keratinocytes for a 2-day period beforeuse. Subsequently, the collagen sponge is turned over and the non-porousside is inoculated with keratinocytes by dispensing drops of inoculumonto various portions of the sponge. The sponge is then incubated withculture medium supplemented with fetal bovine serum for 10 days, againrequiring the lid of the petri dish to be removed every other day forfeeding.

In this example, the petri dish is subject to contamination due to therepeated handling needed for feeding. The delivery of cells also createscontamination risk. There is no way of controlling the amount of mediumthat resides on each side of the sponge because that is dictated by thedensity of the sponge relative to the density of the medium. When thesponge is denser that the medium, it sinks to the bottom of the petridish. When the sponge is facing fibroblast side up, fibroblasts areexposed to the majority of medium in the petri dish. After keratinocyteinoculation, fibroblasts face the bottom of the petri dish exposingcells located towards the center of the sponge to differentconcentrations of medium substrates than those located towards theperimeter of the sponge, since the medium will form gradients towardsthe center of the sponge due to the metabolic activity of the cells. Theproliferation of the cells is a function of how evenly distributed thecells are on the sponge. Therefore, outgrowth varies between each livingskin construct produced in proportion to the variance in the inoculationprocess. If humans are depositing the cells by way of pipetting orsyringe deliver, skin constructs will exhibit a high degree of variancein the initial distribution of cells, even when only one operatorinoculates multiple sponges. More variance can be expected with multipleoperators. Robotic dispensation reduces the variance, but increasescomplexity and does not diminish the exposure to contamination.

The petri dish is very poorly suited to protecting the collagen spongeor helping it retain its desired shape. The collagen sponge must bephysically contacted to lift it and turn it over when the opposite sideof the sponge is inoculated with keratinocytes. This risks damage to thesponge and fibroblasts and again exposes the sponge to contamination.When the culture proliferates, the sponge can contract, as collagen isknown to do when cells grow upon and in it. Thus, the shape of thesponge can change and there is no control over the final shape, aparticularly undesirable characteristic when creating skin constructsthat may be laid side by side on a patient. This leads to an additionalhandling process to cut the sponge into a desired shape with all therisks of sponge damage and contamination present.

Long-term storage of the living skin cannot be done in the petri dishbecause the materials are not compatible with freezing. Therefore, thelid of the petri dish must again be removed and the sponge, now in aweakened condition after having been exposed to culture medium for 20days, must be physically picked up and placed in a cryopreservation bag.Subsequently, the bag must be sealed and filled with cryopreservatives,the process again risking damage to the cells and sponge, and riskingcontamination. This also makes it difficult to perform quality controlin a manner that is inexpensive. Even if non-destructive process controllimits were met during the culture process, such as glucose and oxygenconsumption targets, and those process control evaluations are notcapable of detecting problems that occur once the culture is completeand the sponge is transferred to a cryopreservation bag. If damage orcontamination occurs at that point, it will be expensive to detectbecause the skin will have to be quarantined or a high amount ofdestructive testing will be needed to verify the transfer proceduresused for any given batch of skin were acceptable. Another potentialproblem in process control occurs because the amount of cryoprotectanton each side of the sponge is a function of where the sponge comes toreside in the cryopreservation bag, over which the operator has littlecontrol.

Reconstituting the skin after cryopreservation can be done by removingthe skin from the cryoprotectant bag, placing it in a petri dish, andadding the appropriate medium to reconstitute it, thereby causinghandling and contamination exposure. Subsequently, the sponge needs tobe removed from the petri dish for use, or if not reconstituted at thesite of use (i.e. the hospital), packaged in another bag for shipping.This example demonstrates that it is very difficult to establish tightprocess control for making tissue construct products using the petridish and a new apparatus and method is needed.

Ideally, the apparatus and method would allow protocols that areestablished in the research stages to be relevant in the productionstage. The simplicity of the petri dish is an advantage for thoseperforming research scale cultures. Because the petri dish is compatiblewith typical equipment such as pipettes and incubators, and does notrequire perfusion, those options should remain available as the processis scaled up. In that manner, data generated at the research level wouldremain relevant as scale up occurs, and would not become irrelevant ifthe process were changed completely. Thus, once in the production scale,the device should be capable of operating with continuous perfusion orbatch feeding. Gradient formation in the culture medium should beminimized in both batch fed and perfusion modes of operation. Theimproved device should have an inoculation process that is repeatable,such as the gravitational method commonly used to seed tissue cultureflasks. Cells should come to reside upon the cell attachment matrix asopposed to other surfaces of the device. It should be also possible tocontrol the final shape of the tissue construct in a manner that doesnot expose the construct to damage or contamination. Also, it iscritical that the alternative does not repeatedly expose the tissue tocontamination during the inoculation, feeding, cryopreservation,reconstitution, storage, or shipping stages. Since the possibility ofexposing certain types of cells to medium containing conditioning agentsmay produce better tissue, as in the skin culture example of medium isconditioned by exposure to keratinocytes, the alternative device shouldcontemplate attributes that may cost reduce this process. For example,the use of a membrane to place desired compounds and molecules inproximity of specific areas of the tissue may reduce cost by limitingthe amount of those compounds and molecules needed in the device.

Attempts to address the limitations of the petri dish have beenundertaken, but each attempt only addresses a portion of the problemsand even when combined they do not lead to an alternative that has mostof the desired attributes.

Bell, U.S. Pat. No. 4,435,102, describes a container housing a tissuefor the purpose of assessing the interaction of the tissue and at leastone agent. Although not directed towards overcoming the problems oftissue culture in petri dishes, the art is useful as it provides amethod of controlling the amount of fluid residing above and below thetissue. This is advantageous relative to the inability of the petri dishto maintain predetermined volumes of culture medium above and below theconstruct. Additionally, Bell teaches a method for controlling theshrinkage of the tissue by either constraining the perimeter or allowingit to attach to a membrane for constraint. A method of constraining thecontrolling the shape of the tissue construct by constructing thecollagen matrix in a frame of stainless steel mesh is also disclosed byBell in U.S. Pat. No. 4,485,096.

Bell provides concepts that can be used to address some of the problemsof tissue culture in petri dish. However, by applying them to the skinculture process described above, it can be seen that many problemsremain. Bell does not make it clear how to minimize contaminationpotential throughout the inoculation, feeding, cryopreservation,reconstitution, and preservation stages. Proper oxygenation of thetissue, when cells occupy all sides of the cell attachment matrix, canonly be achieved by perfusing the device with oxygenated medium. This istoo complex and costly for most research environments. Unless the lowercompartment is perfused with oxygen-saturated medium, cells in long-termculture will quickly deplete medium in the lower compartment of oxygen.Because of the low solubility of oxygen in medium relative to thesolubility of needed substrates in medium like glucose, perfusion of thelower compartment to provide oxygen requires a much higher flow ratethan if perfusion just provided substrates. That increases systemcomplexity and cost and subjects cells to a higher rate of shear thanmay be desirable. If perfusion to bring oxygen is not provided once theoxygen in the lower compartment is depleted, the lower portion of thetissue can only obtain oxygen from the medium in the upper compartment.Since the cells on the upper portion of the tissue have first access tooxygen in the medium, the cells on the lower portion of the tissue aresubject to oxygen concentrations that are always reduced relative to thecells on the upper portion of the tissue. Thus, without high flowperfusion, the device is no better than the petri dish for oxygenatingcells at the bottom of the tissue.

In applications where the tissue is to be applied to a patient, such asliving skin, Bell does not provide for a way of preparing the tissuewithout risking damage or increasing contamination risk. If the devicewere to be opened at a hospital for example, the tissue would have to becut out of the frame constraining it. Therefore, since techniques ofcutting the tissue are likely to vary from hospital to hospital, littleprocess control is available. A controlled process would remove thetissue in the same manner each time and lead to superior and consistentoverall tissue quality.

Kemp et al., U.S. Pat. No. 5,536,656 describes controlling shrinkage byway of casting a collagen lattice on an acellular, hydrated collagen gelin contact with a permeable member. For some applications, thisminimizes the need to constrain the collagen about the perimeter. Theuse of an absorbent member in the second, lower compartment in order toprovide a consistent and level physical support for the collagen matrixis disclosed. Advantages are described whereby the absorbent member maycreate diffusional barriers to help retain desirable cell conditioningfactors in proximity of the tissue. However, that same characteristiccan limit transport of desired molecules and compounds to the tissuefrom the surrounding medium. Both the permeable membrane and theabsorbent member can act to prevent inoculation of two sides of a cellattachment matrix because those members block cells from reaching thecell attachment matrix. Importantly, this is not a closed system and therisk of contamination is not diminished relative to the petri dish, andmay actually be increased as two open compartments need to bemanipulated. Furthermore, oxygenation of the culture is limited todiffusion of oxygen from the upper liquid/gas interface. The device doesnot lend itself to process control during scale up since it is notpossible to measure the medium for indicators such as oxygen and glucosewithout taking individual samples from each device.

Peterson et al., U.S. Pat. No. 6,121,042, discloses an apparatus andmethod for seeding and culturing three-dimensional tissue constructs andcreating a dynamic environment, placing mammalian cells under simulatedin vivo conditions resulting in tissue that is more likely to displaythe biochemical, physical, and structural properties of native tissuesthan tissue cultured in a petri dish. The apparatus and methods utilizea variety of methods for physically moving the tissue. Magnetic axialloading and mechanical axial loading of the tissue by way of a piston,bellows, and flexible diaphragm, and pressure cycling the environmentare described. The system is overly complex for tissue that isfunctionally adequate without being physically placed in tension. Thus,at the research scale, the complexity and cost are prohibitive andunnecessary for many applications. Even if the tissue loading elementsare eliminated from the treatment chamber, the system is still toocomplex for research applications as it relies on pumps and otherperfusion support mechanisms. It does not make it clear how to inoculatethe tissue support matrix in a manner that achieves repeatable, uniformseeding of the type needed for applications like the production ofliving skin. It also does not allow removal of the construct from itsconstraints without risking contamination of the treatment chamber anddoes not indicate how to prevent damage to the construct during theremoval process.

The focus is on ligaments, in which an improvement relative to the petridish is attained due to the capability of physically stressing theligament by altering it dimensionally. In this manner the ligament iscultured under conditions more representative of those found in vivo.However, whether or not the apparatus and method are applied toligaments or some other tissue such as skin, many limitations of thepetri dish remain. There is no ability to vary the cross-sectional areaof fluid normal to the plane of the construct, remove trapped gas in anon-perfused system, alter the diffusional distance for gaseouscommunication with the tissue during culture, adequately oxygenate andfeed the culture in the non-perfused state, direct cells to theappropriate location during seeding, make use of centrifugal force as amethod of seeding a cell attachment matrix, control the final shape oftissue construct while retaining a closed system, allowing control ofpredetermined molecules and compounds present in proximity of thetissue. Furthermore, the apparatus and method does not contemplate theneed to protect the bioreactor housing from damage duringcryopreservation if the housing is comprised of a gas permeablematerial, capable of providing passive or non-passive gas transfer toand from the culture, but not entirely compatible with cryopreservationconditions. Also, the use of standard laboratory pipettes for liquidhandling in a manner that minimizes contamination is not contemplated.The apparatus and methods are also complex and eliminate the mostdesirable attribute of the petri dish, which is its simplicity.

SUMMARY OF THE INVENTION

The present invention provides methods and apparatus for inoculating,culturing, cryopreserving, storing, reconstituting, and shipping tissueconstructs in a simple manner that minimizes contamination risk.

One inventive aspect of the present invention involves the use of aflexible tissue construct bioreactor housing that can change shapewithout allowing contamination. The ability for the housing to changeshape allows a variety of benefits to accrue. Changing the shape of thehousing can be done to locate inoculum entirely over the target seedingarea during gravitational seeding and allow the optimal amount ofinoculum to reside above the cell attachment matrix during gravitationalseeding and subsequently the housing volume can again change toaccommodate the optimal medium volume residing in proximity of thetissue, alter the cross-sectional area so that the velocity of fluid inproximity of the tissue can be changed without altering the overallperfusion rate, relieve pressure increases that may result as mediumloses its gas carrying capacity during a temperature increase when batchfeeding is utilized, remove gas that comes to reside in the bioreactorwithout having to displace it with medium and potentially diluteimportant cell produced conditioning agents, change the volume of spacesurrounding the tissue in order to accommodate the addition of theoptimal amount of cyropreservative, change in volume of spacesurrounding the tissue in order to accommodate the addition of theoptimal amount of reconstitution medium, drive an internal die throughthe construct to cut it to a desired shape, or allow microscopic viewingof the tissue.

When the housing of the bioreactor is gas permeable in addition to beingflexible, moving the gas permeable housing closer or farther away fromthe tissue construct will allow oxygen tension to be altered at the celllocation. In this manner, altering oxygen tension can be doneindependent of the ambient surroundings, substantially simplificationrelative to other tissue construct devices. Another unique advantage canbe obtained if the moisture vapor transmission rate differs acrossseparate surfaces of the bioreactor housing. This allows more resolutionin controlling the osmotic conditions in proximity of the cells due toevaporation. This is particularly beneficial in a batch fed process,where there is no replacement of medium for fixed intervals of time.

Another inventive aspect of the present invention is the ability toinoculate and culture cells on a cell attachment matrix residing withina flexible housing in a batch fed manner that retains the simplicity ofthe petri dish, but is superior in terms of minimizing contaminationrisk, improving inoculation distribution, reducing gradient formation,and controlling oxygen tension. The tissue construct resides upon asupport matrix that places it in a position that allows a desired volumeof fluid to reside around it. Also, resolution in oxygen tension controlis available by moving the gas permeable walls closer or farther awayfrom the cells while retaining the same amount of medium in thebioreactor, thereby leaving the concentration of important cell secretedconditioning factors unaltered.

Another inventive aspect of the present invention is the ability tocompartmentalize the bioreactor. This has benefits including allowingeach side of a tissue or cell attachment matrix to be subjected to adifferent medium conditions, allowing inoculation of different celltypes onto different surfaces, and exposing different areas of the cellattachment matrix to different growth conditions such as differentoxygen tension or different glucose concentrations.

Another inventive aspect of the present invention is the ability toallow cells of a different type be co-cultured, physically separated bya semi-permeable membrane, in a manner that includes the attributes ofimproved oxygen tension control, better seeding distribution duringinoculation, and reduced gradient formation.

Another inventive aspect of the present invention is the ability to cutthe tissue to a desired shape without need to expose the tissue tocontamination by opening the bioreactor.

Another inventive aspect of the present invention is the ability toinoculate one or two sides of a cell attachment matrix by intermittentlypositioning the bioreactor in a vertical and horizontal position inorder to allow a well mixed inoculum to deposit cells in a welldistributed pattern upon a cell attachment matrix, and create optimalculture conditions by allowing the tissue to receive medium in perfusionor batch modes.

Another inventive aspect of the present invention is the ability to seedcells into a cell attachment matrix by the use of centrifugal force.

Another inventive aspect of the present invention is the ability toaccess a septum by way of a needle while protecting both the needle andseptum from exposure to contamination before, during, and after needlepenetration.

Another inventive aspect of the present invention is the ability toaccess a flexible container with a pipette in a manner that preventscontaminants from entering the container while the pipette is engaged inthe access port of the container.

It is yet another inventive aspect of the present invention to enclosethe tissue construct bioreactor in a package that prevents damage to thetissue during cryopreservation even if the housing to the bioreactor isdamaged during the process.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an embodiment of a tissue construct bioreactor housing a cellattachment matrix.

FIG. 2A, FIG. 2B, and FIG. 2C disclose an example of an embodiment inwhich the tissue construct bioreactor is configured to be shipped,sterilized, and stored in a minimum volume condition, expand in volumeat the onset of the culture process, and change in volume during theculture process.

FIG. 3A and FIG. 3B disclose an example of an embodiment of thisinvention in which the walls of the tissue construct bioreactor arecomprised of a rigid material. The tissue construct bioreactor isconfigured to change in volume during the culture process withoutbreaching sterility.

FIG. 4A and FIG. 4B disclose a configuration of an embodiment where gasis removed from tissue construct bioreactor without displacing it withliquid.

FIG. 5A, FIG. 5B, FIG. 5C, and FIG. 5D show an embodiment of a tissueconstruct bioreactor configured to allow cells to be seeded onto oneside of the cell attachment matrix, and subsequently allow a desiredamount of medium to reside on each side of the cell attachment matrix bypositioning the tissue construct bioreactor in a vertical position.

FIG. 6A and FIG. 6B show an embodiment for inoculating both sides of acell attachment matrix and allowing a predetermined amount of medium toreside on each side of the cell attachment matrix.

FIG. 7 shows one embodiment of a tissue construct bioreactor that allowsa desired amount of medium or fluid to reside on each side of a cellattachment matrix.

FIG. 8 shows an embodiment of a tissue construct bioreactor in which acell attachment matrix is held in a desired position by a frame.

FIG. 9 shows an embodiment of a compartmentalized tissue constructbioreactor configured to allow independent access to each compartment.

FIG. 10 shows an embodiment in which a tissue construct bioreactor iscompartmentalized by a membrane.

FIG. 11 shows an embodiment of a tissue construct bioreactor configuredto cut a cell attachment matrix to a desired shape without any need tofirst remove the tissue construct from the tissue construct bioreactor.

FIG. 12 shows a cutaway view of an embodiment in which a cutting die isconfigured to cut a cell attachment matrix into multiple sections.

FIG. 13A and FIG. 13B show one example of an embodiment of a tissueconstruct bioreactor comprising two compartments and a cutting dieuseful for separating the desired portion of the cell attachment matrixfrom the undesired portion.

FIG. 14A and FIG. 14B show cross-sectional views of an embodiment inwhich a tissue construct bioreactor is configured to allow a cellattachment matrix to be removed.

FIG. 15A and FIG. 15B show the inoculation and feeding stages of anembodiment of a tissue construct bioreactor in which a cell attachmentmatrix is held in a desired position by a frame.

In FIG. 16, a non-flexible outer housing mates with a flexible tissueconstruct bioreactor wall at predetermined points to prevent it fromcollapsing.

FIG. 17 shows a tissue construct bioreactor with walls constrained in aplanar state by an outer housing.

FIG. 18 shows another configuration for maintaining a relativelyparallel geometric relationship and fixed minimum distance between acell attachment matrix and the walls of a flexible tissue constructbioreactor.

FIG. 19 shows a configuration in which a grid is used to maintain arelatively parallel relationship and fixed minimum distance between acell attachment matrix and a flexible tissue construct bioreactor wall.

FIG. 20 shows a tissue construct bioreactor configured to maintain acell attachment matrix from contacting the walls of a tissue constructbioreactor and to allow the cell attachment matrix to be cut to adesired shape.

FIG. 21A and FIG. 21B show a method of inoculating one side of a cellattachment matrix residing within a compartmentalized tissue constructbioreactor.

FIG. 22A and FIG. 22B show a method of inoculating two sides of a cellattachment matrix residing within a compartmentalized tissue constructbioreactor.

FIG. 23A, FIG. 23B, and FIG. 23C shows a gravitational method ofinoculating two sides of a cell attachment matrix residing within acompartmentalized tissue construct bioreactor.

FIG. 24A and FIG. 24B show an embodiment for inoculating one side, andfeeding both sides, of a cell attachment matrix.

FIG. 25 shows an embodiment of a compartmentalized tissue constructbioreactor configured for needle access.

FIG. 26A, FIG. 26B, and FIG. 26C show an embodiment for needle accessthat minimizes exposure to contamination.

FIG. 27A, FIG. 27B, FIG. 27C, and FIG. 27D disclose configurations of anembodiment for pipette access in a liquid tight manner that allowsnon-sterile surfaces to reside in areas other than directly above accessports.

FIG. 28 shows an embodiment of a tissue construct bioreactor adapted forcryopreservation in the case where the material selected for the tissueconstruct bioreactor walls is not compatible with cryopreservation, orwhen a redundant seal is desired.

REFERENCE NUMERALS IN DRAWINGS

-   10 tissue construct bioreactor-   20 cell attachment matrix-   25 lower cell attachment matrix-   30 tissue construct bioreactor walls-   35 wall seal-   37 clamp-   38 constraining clamp-   40 access port-   42 thin walled access opening-   43 pipette tip-   44 pipette-   45 void volume-   46 fluid access channel-   47 pipette stop-   48 threads-   49 flexible tube-   51 cap-   60 gas-   70 cell culture medium-   80 septum-   90 syringe needle-   100 syringe-   110 syringe flow direction-   120 wall direction arrow-   130 cryopreservation enclosure-   140 gas compartment-   145 gas compartment access port-   150 sterile gas filter-   160 gas compartment spacer-   170 cutting die-   180 matrix holder-   190 legs-   200 frame-   202 membrane-   220 inoculum-   230 compartmentalized tissue construct bioreactor-   240 first compartment-   250 second compartment-   260 bioreactor lid-   270 bioreactor body-   280 lid seal-   290 moveable latch-   310 vertical walls-   330 outer housing-   340 boss-   350 pocket-   360 spacer projections-   370 grid-   380 access needles-   390 access needle protection septum

DETAILED DESCRIPTION OF THE INVENTION

The following embodiments of the present invention will be described inthe context of an apparatus and method for seeding a cell attachmentmatrix with tissue, culturing, preserving, reconstituting, altering thegeometric shape of, shipping, and removing the tissue. Those skilled inthe art will recognize that the apparatus and methods are useful forbroader applications including those in which tissue is present withouta cell attachment matrix.

FIG. 1 shows an embodiment of a tissue construct bioreactor 10configured for culture of a tissue construct. Cell attachment matrix 20resides within tissue construct bioreactor 10 for the purpose ofbecoming populated with cells of a desired type. Tissue constructbioreactor walls 30 are liquid impermeable, and closed to the ambientgas. Cell attachment matrix 20 can be any material capable of allowingcells to reside upon it, or within it. For example, it can be comprisedof collagen gel [Yang et al., 1981] cellulose sponge [Leighton et al.,1951], collagen coated cellulose sponge [Leighton et al., 1968], gelatinsponge, Gelfoam™ [Sorour et al., 1975], collagen sponge cross-linked toform one side into a non-porous skin [Eisenburg 2000], porous polyvinylformal resin [Tun et al. 2000], nylon, dacron, polystyrene,polypropylene, polyacrylates, polyvinyl compounds, polycarbonate,polytetrabluorethylene, thermanox, nitrocellulose [Naughton et al.,1999], microporous membrane, semipermeable membrane, or any othermaterial that has been used for tissue culture. Preferably, cellattachment matrix 20 resides within tissue construct bioreactor 10 priorto sterilization. That minimizes contamination risk associated withaseptic insertion. However, depending on the characteristics of cellattachment matrix 20, such as compatibility with various sterilizationmethods, aseptic insertion may need to occur after tissue constructbioreactor 10 has been sterilized or it may be formed in place.

If at least a portion of one tissue construct bioreactor wall 30 is acomprised of a liquid impermeable, gas permeable material, gas candiffuse into and out of tissue construct bioreactor 10. That allowscells to obtain gas exchange independent of the rate of cell culturemedium delivery. In many perfused bioreactors, medium is oxygenatedprior to delivery to the bioreactor. The low solubility of oxygen inmedium, when compared to substrates such as glucose and essential aminoacids, requires perfusion rates be dictated by the oxygen demand of thetissue construct. However, by making a portion of the bioreactor housinggas permeable, perfusion of oxygenated medium into the bioreactor tosupport cellular demand can be avoided. When oxygen is delivered by wayof the gas permeable bioreactor housing, several factors affect thedelivery rate of oxygen to the cell mass. They include the surface areaof gas permeable material available for transfer, permeability of thegas permeable material, the degree of parallelism between cellattachment matrix 20 and gas permeable tissue construct wall 30, thedistance between gas permeable tissue construct wall 30 and the cellmass, and if gas and liquid resides in tissue construct bioreactor 10,the distance between the cell mass and the gas/liquid interface.

The amount of cell culture medium residing in gas permeable tissueconstruct bioreactor 10 has an effect on bioreactor geometry and gasexchange. If cell culture medium is delivered in a batch fed mode, thegeometry of tissue construct bioreactor 10 should take intoconsideration the need to provide the tissue construct with enough cellculture medium to sustain the culture for a predetermined period oftime. When feeding is done manually, typically at least 24 hours betweencell culture medium exchanges is most convenient for operators.Additional cell culture medium capacity will make feeding less frequent,but should be balanced against any negative impact on gas exchange ifthe distance between the cell mass and the oxygen source increases as aresult of increasing cell culture medium capacity.

Since petri dish culture of tissue constructs is common, the petri dishgeometry provides a good reference for batch fed tissue constructbioreactor design. Typically, in petri dish culture, the tissueconstruct not the same dimension as the petri dish and therefore not allof the cell culture medium resides directly above the tissue construct.Thus, the distance from the tissue construct to the gas/liquid interfaceis less than it would be if all of the liquid resided directly above thetissue construct. As a result, when the volume of medium is fixed,oxygen is required to travel farther and surface area for gas transferis reduced as the configuration is altered to allow more medium toreside directly above the tissue construct. For example, if a 6 cm×6 cmcell attachment matrix resides in a 145 mm diameter petri dish (165 cm2surface area) containing 50 ml of cell culture medium, the height ofliquid that resides in the petri dish is approximately 3.0 mm. However,if the bottom of the petri dish were the same size and shape as the cellattachment matrix, thereby allowing liquid to reside entirely over thecell attachment matrix, the height of liquid above the cell attachmentmatrix would more than double to 7.2 mm.

Despite increasing the distance from cells to the gas source andlimiting the surface area available for gas transfer, there areadvantages to configurations in which a majority of the cell culturemedium resides above the tissue construct. For example, duringgravitational seeding if inoculum resides directly over the cellattachment matrix cells will deposit upon the cell attachment matrix,and not upon other areas of the bioreactor as commonly occurs with petridish culture. Minimizing the amount of cell attachment onto areas of thebioreactor other than the cell attachment matrix is useful forapplications in which cells that deposit and attach to surfaces of thetissue construct bioreactor, other that the cell attachment matrix, havea detrimental effect on the culture as described in U.S. Pat. No.5,266,480. For applications that are typically performed in petridishes, the tissue construct bioreactor can be configured to allownearly all of the inoculum to reside directly over the cell attachmentmatrix.

In the case where the tissue construct bioreactor walls are not gaspermeable, and perfusion is not present, configuring the tissueconstruct bioreactor so that it can contain a gas/liquid interface willallow gas exchange in a batch fed tissue construct bioreactor. The gasshould be in sterile communication with a source of gas residing outsideof the bioreactor. Gaseous communication by way of a gas permeablesterile filter will prevent contamination. The gas/liquid interface actsto increase surface area for gas transfer relative to a non-gaspermeable tissue construct bioreactor completely filled with cellculture medium.

If gas permeable walls are desired, a variety of gas permeable materialscan be used. In general, suitable gas permeable materials are flexible.If the tissue construct bioreactor is comprised of flexible materials,there can be geometric benefits in addition to gas exchange as describedherein. The best choice of material for gas exchange depends on thespecific culture application. As a general guideline, the gaspermeability of a given material should be considered in addition to theinteraction of the material with either cells or protein structures.Liquid impermeable films of an equivalent thickness, exposed toequivalent cellular oxygen demand, will establish various steady stateoxygen tensions in the cell culture medium. For example, fluorinatedethylene polymers, silicone, Teflon, and silicone polycarbonatecopolymers will establish higher oxygen tension in the tissue constructbioreactor than will polyethylene, polycarbonate, polypropylene,polysulfone, or polypropylene polymers. In some cases, such as when costcan be reduced or a hard shell housing can make handling easier, it maybe desirable to only have a portion of the tissue construct bioreactorgas permeable. This is acceptable, provided that adequate gas exchangeis achieved. In other cases, it may be desirable to have more than onetype of gas permeable material present, such as the case where differentgas transfer rates are desired. For example, when both the upper andlower tissue construct bioreactor walls are gas permeable, it may bedesirable to minimize evaporation at the location closet to the cellmass due to the potential for increased local osmolarity. If that werethe case in the configuration described in FIG. 1, lower tissueconstruct bioreactor wall 30 could be a material with a lower moisturevapor transmission rate than the material selected for the upper tissueconstruct bioreactor wall 30.

Constructing the tissue construct bioreactor so that is can changevolume without a breach in sterility can be beneficial. Benefits relatedto altering the volume of the tissue construct bioreactor include theability to reduce the amount of packaging material, cost oftransportation, and the use of inventory space. For example, packagingmaterial can be reduced if the tissue construct bioreactor initiallyresides in a minimum volume state when place into a sterility bag, issubsequently removed from the sterility bag for use, and then expands involume in response to fluids added during the cell culture process. Thesmaller sized sterility bag reduces the cost of packaging material,shipping, sterilization, and storage.

Furthermore, constructing the tissue construct bioreactor with theability to change volume while remaining a closed system allowsvariability in the volume of gas and/or liquid that can reside in thetissue construct bioreactor at any given time. A wide variety ofcombinations of gas and liquid volumes can be retained in the tissueconstruct bioreactor.

Altering the distance between the tissue construct and the walls of thetissue construct bioreactor at some point after the culture process hasbeen initiated can be beneficial. For example, bringing a wall of thebioreactor closer to the tissue construct at some point during aperfused culture may helpful in generating a well-mixed flow profile oraltered velocity profile. Other beneficial reasons may be present, suchas when the optimal volume of inoculum differs from the optimal volumeof cell culture medium contained in the tissue construct bioreactorduring the culture phase, when an increase in the gas exchange demandsof a culture requires a gas permeable wall to get closer to the culture,when the oxygen tension that is optimal for cells needs to be alteredwithout the ability to change the oxygen tension of the ambientsurrounding, when microscopic viewing is needed and the focal length ofthe microscope at the desired magnification requires moving either theupper wall or lower wall closer to the tissue construct depending onwhat surface of the tissue construct is to be viewed, or when theappropriate volume of cryoprotectant differs from the appropriate amountof medium for culture.

FIG. 2A and FIG. 2B disclose an example of an embodiment in which tissueconstruct bioreactor 10 is configured to be shipped, sterilized, andstored in a minimum volume condition, expand in volume at the onset ofthe culture process, and change in volume during the culture process. Toattain these objectives, at least one wall of tissue constructbioreactor 10 is flexible. FIG. 2A shows tissue construct bioreactor 10in a state of minimum volume prior to the onset of the culture process.FIG. 2B shows tissue construct bioreactor 10 in a state of maximumvolume, having been altered in volume as flexible tissue constructbioreactor wall 30 expands due to the addition of inoculum 220. FIG. 2Cshows tissue construct bioreactor 10 in a state of reduced volumerelative to that shown in FIG. 2B such as may be the case when theoptimal volume of cell culture medium 70 is less than the volume ofinoculum 220. Access into tissue construct bioreactor 10 can be madethrough a single access port 40. In this embodiment, tissue constructbioreactor walls 30 are comprised of flexible materials. Inoculum 220can be delivered in a sterile manner by penetrating access port 40 witha needle, thereby causing tissue construct bioreactor 10 to expand involume. Likewise, after cells of inoculum 220 have deposited onto cellattachment matrix 20, residual inoculum can be removed by a needle,thereby causing tissue construct bioreactor 10 to contract in volume.

The volume of the tissue construct bioreactor can be altered in asterile manner without the use of flexible walls. FIG. 3A and FIG. 3Bdisclose an example of an embodiment of this invention in which tissueconstruct bioreactor 10 changes volume during the culture process. Inthis configuration, the walls of tissue construct bioreactor 10 arecomprised of rigid materials. The upper tissue construct bioreactor wall30 can be moved either towards or away from cell attachment matrix 20 atsome predetermined point in the culture. Wall seal 35 between the uppertissue construct bioreactor wall 30 and the lower tissue constructbioreactor wall 30 prevents contamination or leakage. When reducingvolume, such as when displacing inoculum 220 with cell culture medium 70post cell attachment to cell attachment matrix 20, fluid can bedisplaced in a sterile manner into a liquid handling device such assyringe 100, or into a reservoir when perfusion is employed.

Minimizing the volume of gas residing within the tissue constructbioreactor can be beneficial in tissue construct applications in whichcells should not come into direct contact with gas/liquid interfaces dueto resulting detrimental effects such as those related to shear, or whencells, such as keratinocytes, can differentiate when exposed to gas. Inbioreactors with a fixed volume, unwanted gas can become present duringthe culture even if it is not present at the onset of the culture. Forexample, this can occur if the cell culture medium rises in temperature,thereby experiencing a reduction in its gas carrying capacity. FIG. 4Aand FIG. 4B disclose a configuration of an embodiment where gas 60 isremoved from tissue construct bioreactor 10 without displacing it withliquid. Tissue construct bioreactor 10, which is configured to change involume, retains only the initial volume of cell culture medium 70 aftergas 60 is removed. Septum 80 is penetrated by syringe needle 90. Thebeginning of the process is shown in FIG. 4A, where syringe 100 is showndrawing gas 60 in the direction indicated by syringe flow directionarrow 110. Flexible tissue construct bioreactor wall 30 begins to movein the direction shown by wall direction arrow 120 as gas 60 is removeddue to the pressure differential created across flexible tissueconstruct bioreactor wall 30. In FIG. 4B flexible tissue constructbioreactor wall 30 has collapsed to occupy the space in which gas 60previously resided. If tissue construct bioreactor 10 could not changevolume, and a single access port were desired, cell culture medium wouldhave to be added to displace the gas. Thus both gas and cell culturemedium would need to move simultaneously through the single access port,leaving the port open to contamination during this procedure. In a fixedvolume tissue construct bioreactor, the use of two ports adapted forhermetic interface with liquid handling equipment, such as by the use ofluer tapers, would minimize contamination. However, the need to addliquid in order to displace gas would change the quantity and perhapsthe concentration of important metabolites.

Even in applications in which the presence of gas is acceptable from abiological standpoint, detrimental operational effects can occur if gasvolume increases during use. A pressure increase within a bioreactor canoccur when cell culture medium loses its gas carrying capacity if thetissue construct bioreactor is not free to expand in volume. Pressurewill then seek to equilibrate with ambient conditions when thebioreactor access port, or ports, are accessed. This will increase thecontamination risk if pressurized fluids, such as cell culture medium,are displaced from the bioreactor when a port is accessed. A flexibletissue construct bioreactor wall will alleviate this condition since itcan expand to accommodate gas exiting the cell culture medium, therebyreducing the internal pressure relative to a bioreactor withnon-flexible walls. Eventually, the gas permeable portion of the tissueconstruct bioreactor will allow pressure to equilibrate with ambientconditions by diffusion.

In a typical petri dish culture, the cell attachment matrix will settleto a given location determined by its specific gravity, and the amountof cell culture medium that resides on each side of the tissue constructis determined the location at which the cell attachment matrix comes toreside. Increasing control over the volume of medium residing on eachside of the tissue construct can enhance repeatability in the cultureprocess. The use of flexible tissue construct bioreactor walls can beadvantageous when configuring the tissue construct bioreactor with thecapability to allow a desired amount of cell culture medium to reside oneach side of the tissue construct.

FIG. 5 shows an embodiment of a tissue construct bioreactor configuredto allow cells to be seeded onto one side of the cell attachment matrix,and subsequently allow a desired amount of medium to reside on each sideof the cell attachment matrix by positioning the tissue constructbioreactor in a vertical position. Cell attachment matrix 20 is capturedon one edge by clamp 37, which is secured to tissue construct bioreactor10. In FIG. 5A, tissue construct bioreactor 10 is positionedhorizontally for gravitationally seeding cell attachment matrix 20. Cellattachment matrix 20 resides in proximity of the lower interior surfaceof tissue construct bioreactor wall 30 due to its specific gravity. Ifnecessary, a weight can be attached to cell attachment matrix 20 toallow it to reach this lower surface by gravitational force. The weightshould be positioned in a manner that maximizes the available surfacefor cell deposit onto cell attachment matrix 20. When cell attachmentmatrix 20 resides in proximity to the lower surface, the amount ofinoculum that can reside between cell attachment matrix 20 and the lowerinterior surface is minimized, thereby maximizing cell deposit on cellattachment matrix 20. When minimizing the amount of cells that attach tosurfaces other than cell attachment matrix 20 is desired, structuringthe walls of tissue construct bioreactor 10 with hydrophobic materialsis beneficial. Constraining clamp 38 limits the location of inoculum 220such that the vast majority of inoculum 220 resides above cellattachment matrix 20 and cells settle onto the exposed face of cellattachment matrix 20. Alternatively, dimensioning the wall of tissueconstruct bioreactor 10 such that the vast majority of inoculum 220resides above cell attachment matrix 20 will help direct cells to thesurface of cell attachment matrix 20, as best shown in FIG. 5B.

The tissue construct bioreactor and inoculation process should typicallybe designed with the objective of achieving a relatively evendistribution of cells across the desired surface of the cell attachmentmatrix, while minimizing the deposit of cells onto other surfaces. Thus,positioning the cell attachment matrix to reside in a planar state willenhance seeding uniformity. When the tissue construct bioreactor is inthe horizontal position, a well mixed cell suspension placed above thecell attachment matrix will allow cells to gravitationally settle uponthe matrix in a well distributed pattern. In some applications, it maybe desirable for the contact force that cells make with the cellattachment matrix during inoculation to exceed that caused by gravity inorder to drive them into cell attachment matrix 20. In that case,placing the mixed suspension in the tissue construct bioreactor, andthen centrifuging the bioreactor in a direction and at a velocity thatdrives cells into the cell attachment matrix can achieve that purpose.The same accelerated force typically used to re-suspend the specifictype of cells in the application should be the initial target force forseeding so that the cells remain undamaged. Proper physical support forcell attachment matrix 20 should be present so that it remains in arelatively planar state. Iterations to the rotation rate to achieve themost desirable seeding pattern may be necessary, depending on thespecific combinations of materials, cell type, and desired degree ofpenetration.

After cells have attached to cell attachment matrix 20, tissue constructbioreactor 10 is oriented in a vertical position such that clamp 37resides at the upper position as shown in FIG. 5C. To minimize gradientformation, flexible tissue construct bioreactor walls 30 are constrainedby outer housing 330 such that they are substantially parallel to cellattachment matrix 20. For best control over the volume of medium thatresides on each side of cell attachment matrix 20, cell attachmentmatrix 20 hangs in a relatively planar state under the force of its ownweight, or an attached weight if necessary. The volume of cell culturemedium 70 residing on either side of cell attachment matrix 20 can becontrolled by orienting the constrained edge of cell attachment matrix20 in a manner that positions it a desired distance from tissueconstruct bioreactor walls 30. For example, in FIG. 5C the distancebetween the each surface of cell attachment matrix 20 and tissueconstruct bioreactor wall 30 is relatively equal. In FIG. 5D, theconstrained edge of cell attachment matrix 20 has been shifted towardsthe leftmost wall of outer housing 330 by rotating the perimeter oftissue construct bioreactor 10 in a counter clockwise direction. Postrotation, the volume of cell culture medium 70 residing to the left ofcell attachment matrix 20 is less than that residing to the right ofcell attachment matrix 20. Creating a non-symmetric position may beuseful for a variety of reasons such as when only one of tissueconstruct bioreactor walls 30 is gas permeable and positioning one faceof cell attachment matrix 20 closer to that gas permeable tissueconstruct bioreactor wall 30 enhances gas exchange of the cells residingon that face.

Rotating the perimeter of tissue construct bioreactor 10 is madepossible due to the flexible material comprising tissue constructbioreactor walls 30. A portion of tissue construct bioreactor wall 30can be comprised of rigid material, provided that the rigid portion doesnot preclude positioning cell attachment matrix 20 in the desiredlocation. At a minimum, the perimeter section comprising the upper andlower portion of the tissue construct bioreactor walls, when oriented inthe vertical position, should be flexible so that the cell attachmentmatrix is capable of moving into the desired location within the tissueconstruct bioreactor.

FIG. 6 shows a tissue construct bioreactor embodiment for inoculatingboth sides of the cell attachment matrix and allowing a predeterminedamount of medium to reside on each side of the cell attachment matrix.Cell attachment matrix 20 is captured on one edge by clamp 37, which issecured to a flexible tissue construct bioreactor wall 30 section oftissue construct bioreactor 10. Those skilled in the art will recognizethat any method used to secure one edge of cell attachment matrix 20 isacceptable. In FIG. 6A, tissue construct bioreactor 10 resides in ahorizontal position such that inoculum 220 gravitationally seeds ontothe uppermost face of cell attachment matrix 20, which resides inproximity of the lower interior surface of tissue construct bioreactor10 due to its specific gravity. If necessary, cell attachment matrix 20can be weighted to allow it to reach this lower interior surface bygravitational force. The weight should be positioned in a manner thatmaximizes the available surface for cell deposit onto cell attachmentmatrix 20. Residing in proximity to the lower surface minimizes theamount of inoculum that can reside between cell attachment matrix 20 andthe lower interior surface, thereby maximizing cell deposit onto thetrapped upper face of cell attachment matrix 20. After cells haveattached to cell attachment matrix 20, tissue construct bioreactor 10 isturned over and clamp 37 is positioned such that cell attachment matrix20 is in proximity of the lower interior surface of tissue constructbioreactor 10 as best shown in FIG. 6B. Inoculum 220 containing the typeof cells desired is placed into tissue construct bioreactor 10,preferably such that the vast majority of it resides above cellattachment matrix 20. Cells settle by gravity, or alternatively bycentrifugation, onto the exposed surface of cell attachment matrix 20.After cells have attached to cell attachment matrix 20, tissue constructbioreactor 10 is oriented in a vertical position such that clamp 37resides at the upper position as previously described and shown in FIG.5B. Adjusting the volume of cell culture medium residing on each side ofthe cell attachment matrix can be accomplished as previously describedand shown in FIG. 5C.

FIG. 7 shows another embodiment a compartmentalized tissue constructbioreactor 230 that allows a desired amount of medium or fluid to resideon each side of cell attachment matrix 20. Matrix holder 180 holds cellattachment matrix 20 in a desired location. In this configuration, apredetermined volume of cell culture medium 70 resides below cellattachment matrix 20 in second compartment 250. Matrix holder 180 haslegs 190 dimensioned in height to allow the predetermined volume of cellculture medium 70 to reside below cell attachment matrix 20. Matrixholder 180 should be designed with consideration given to the mechanicalstrength of cell attachment matrix 20 when wet. If cell attachmentmatrix 20 is not capable of supporting its own weight, matrix holder 180makes contact with the lower surface of cell attachment matrix 20.Matrix holder 180 can be comprised of a variety of materials andconfigurations that allow cell culture medium 70, or desired compoundsof cell culture medium 70, to contact cell attachment matrix 20. Supportconfigurations include microporous membranes, semi-permeable membranes,support grids, and open weave mesh. Unless membranes are used forsupport, matrix holder 180 should be configured such that minimalcontact with cell attachment matrix 20 exists, thereby permittingrelatively unencumbered delivery of nutrients and removal of wasteproducts. For example, an open diamond weave mesh such as that with ½inch openings (Catalogue # 18-157-50 Nalle Plastics, Austin Tex.) iscapable of providing adequate structural support and allowing relativelyunencumbered mass transfer when cell attachment matrix 20 is comprisedof 0.020 inch thick bovine collagen. If microporous or semipermeablemembranes are used, additional physical support structures may or maynot be needed depending on the capacity of the membrane to retain cellattachment matrix 20 in a relatively planar state. For example,cellulose membranes swell when wet. When sealed around their perimeterin the dry state, and then wetted, the swelling causes slack in themembrane thereby reducing its capacity to maintain a planar position.However other membranes, such as microporous polycarbonate membranes,retain dimensional stability when wetted. If the purpose of usingmembranes is to make compounds and molecules of a predetermined sizeavailable to the tissue construct by physically preventing undesiredcompounds and molecules of a predetermined size from moving from thecell culture medium residing in first compartment 240 with that residingin second compartment 250, the perimeter of the membrane must seal tomatrix holder 180 to prevent shunting of undesired compounds andmolecules. In the preferred embodiment, cell attachment matrix 20resides in a substantially horizontal position such that cells cansettle onto the entire surface of cell attachment matrix 20 duringinoculation. Matrix holder vertical walls 310 retain inoculum 220 abovecell attachment matrix 20 in first compartment 240 during seeding. Whentissue construct bioreactor 10 resides in the horizontal position, andthe density of cell attachment matrix 20 exceeds that of cell culturemedium, and cell attachment matrix 20 is not physically constrained,gravity keeps cell attachment matrix 20 located upon matrix holder 180.Gas that may become present directly below cell attachment matrix 20will act to inhibit mass transfer of solutes. Any gas that becomestrapped below cell attachment matrix 20 can be removed by orientingtissue construct bioreactor 10 in a manner such that gas is allowed torise from under cell attachment matrix 20 without permanently dislodgingcell attachment matrix 20 from matrix holder 180. If cell attachmentmatrix 20 is physically constrained to maintain its position upon matrixholder 180, care should be taken to ensure the mechanism acting toconstrain cell attachment matrix 20 does not inhibit uniform seeding,feeding, or gas exchange.

For co-culture applications in which it is desirable to separate cells,two cell attachment matrices can reside in the bioreactor. FIG. 7 showsoptional lower cell attachment matrix 25, useful for co-culture. Whenmatrix holder 180 integrates a membrane in the manner described above,the membrane can be used to retain desirable molecules and compounds inthe presence of cells on one cell attachment matrix without allowingthose molecules and compounds to be available to cells of the other cellattachment matrix. In some cases it may only be desirable to preventcells from migrating from one cell attachment matrix and making physicalcontact with cells of the other cell attachment matrix. Thus, theparticular purpose of the co-culture application will dictate themolecular weight cutoff or micro-porosity of the membrane. As oneexample, in co-culture applications typically performed in commerciallyavailable devices, such as a transwell, microporous membrane with a 0.2to 1.2 micron opening is preferred. In other applications, a lowercutoff may be desirable such as a 10,000 MWCO non-protein bindingmembrane when serum is in proximity of one cell attachment matrix butnot in the other. The type of membrane best suited for a givenapplication will be based upon many variables including size exclusion,cell attachment capacity, protein binding characteristics, sterilizationcompatibility, mass transfer capacity, cost, commercial availability,and biocompatibility. Those not familiar with the best choices for themembrane properties should review literature describing the use ofmembranes in cell culture applications. It should also be noted that theuse of the description cell attachment matrix does not preclude the useof suspension cells. Additionally, the cell attachment matrix may be themembrane and the lower wall of the tissue construct bioreactor or justthe upper and lower sides of the membrane. For example, a commonly usedmaterial for cell culture is polystryene, which if present in the lowertissue construct bioreactor wall 30 would allow the wall to constitute acell attachment matrix for adherent cells to attach to or suspensioncells to reside upon. However, for suspension cells there may be anadvantage when the lower tissue construct wall is comprised of gaspermeable material, thereby allowing better gas transfer of thesuspension culture than if the oxygen were delivered by way of a gasliquid interface above the cells. In this case, for definition purposes,the lower tissue construct wall would also constitute a cell attachmentmatrix. A main advantage that the tissue construct bioreactor brings toco-culture relative to the traditional use of a transwell is thecapacity to remain a closed system and place cells of the lower cellattachment matrix 25 in closer proximity of gas exchange when the lowertissue construct bioreactor wall is comprised of a gas permeablematerial.

FIG. 8 shows an embodiment of tissue construct bioreactor 10 in whichcell attachment matrix 20 is held in a desired position by frame 200. Inthis configuration, frame 200 is configured to allow a defined volume ofcell culture medium 70 to reside below cell attachment matrix 20 and toprevent cell attachment matrix 20 from changing shape during the cultureprocess. The lower surface of cell attachment matrix 20 has unobstructedcontact with cell culture medium 70 when frame 200 is designed to allowmedium to move through it. Unobstructed contact with medium 70 can alsobe attained using the materials and methods described for matrix holder180 of FIG. 7. If cell culture medium 70 is added to tissue constructbioreactor 10 while tissue construct bioreactor 10 resides in ahorizontal position, the design should allow gas to exit from the volumeof space directly below cell attachment matrix 20. This can be achievedby a method of adding cell culture medium 70 until its height exceedsthe height of the lower surface of cell attachment matrix 20, andtemporarily orienting tissue construct bioreactor 10 to a position thatallows gas to move away from the lower surface of cell attachment matrix20. Another method would be to add the medium while tissue constructbioreactor 10 is oriented with cell attachment matrix 20 tilted at anangle that allows gas to exit from the underside of cell attachmentmatrix 20. Tissue construct bioreactor 10 could remain in this positionthroughout the culture, provided cells have attached to cell attachmentmatrix 20.

FIG. 9 shows an embodiment of a compartmentalized tissue constructbioreactor configured to allow independent access to each compartment.Compartmentalized tissue construct bioreactor 230 houses cell attachmentmatrix 20 in a predetermined position and in a manner that creates firstcompartment 240 and second compartment 250. As previously described, ifthe tissue construct bioreactor walls are flexible, one access port canbe used to access each compartment with a needle or a syringe withoutexposure to airborne contaminants. More ports may be desirable dependingthe objectives of medium delivery, which include rates of fluid deliveryand removal, distribution of metabolites, distribution of cells ininoculum, and removal of undesired gas. In this embodiment, tissueconstruct bioreactor walls 30 are flexible, and access ports 40 areconfigured to form a seal with liquid handling equipment.

Each side of cell attachment matrix 20 can contain the same type ofcell, or different types of cells, or one side may have no cells. Eachcompartment can house a different type of medium, or the same type ofmedium. Medium can comprise a liquid containing soluble nutrientsubstrates, or compounds used to elicit a response in the construct,such as when challenging a skin construct with an allergen, or fluidsuch as gas.

FIG. 10 shows an embodiment in which compartmentalized tissue constructbioreactor 230 is compartmentalized by membrane 202. It is advantageousrelative to the embodiment described in FIG. 7 when it is desirable toorient the bioreactor in non-horizontal position or when completeisolation of the compartments by membrane 202 is desired. Membrane 202can function as the cell attachment matrix. Use of membrane 202, asdescribed previously, allows specific environmental conditions to beestablished for cells that grow in suspension those that are adherent.For example, when a particular cell type is secreting a cytokine thatconditions the cell culture medium favorably, the use of a membrane thatretains the cytokine in proximity of the cell could be beneficial. Asanother example, if TGF beta is introduced into one compartment, asemi-permeable membrane with a molecular weight cutoff less than that ofthe TGF beta can be used to retain the TGF beta in that compartment. Inother cases, the transfer characteristics of a membrane are based on thedesire to allow transfer from one compartment to another, such as whenTGF beta is produced by cells in one compartment and used to stimulateactivity of cells in the other compartment. In this case, the membranesmolecular weight cutoff would need to exceed the size of TGF beta. Forexample, if TGF beta were about 25,000 MW, a membrane of 30,000 MWCOwould allow TGF beta to cross the membrane. The use of microporousmembranes may also be desired when evaluating the absorption of a drugintroduced into the compartment in which cells reside confluently uponthe membrane. Monitoring the other compartment for presence of the drugcan indicate the capacity of the drug to diffuse through the cell mass.Another application could be the culture of skin on one side of themembrane and exposure of the skin to selective compounds to monitor skinreaction as an alternative to animal testing. The use of semi-permeableor microporous membranes can also be useful for co-culture of cells aspreviously described. In these applications, the membrane can functionas both the cell attachment matrix and a barrier to cells, molecules andcompounds of a predetermined size. Optional lower cell attachment matrix25, which in its most simple form may be a portion of lower tissueconstruct bioreactor wall 30, allows cells to reside in secondcompartment 250.

Cutting a tissue construct to a specific shape after culture can bedesirable when the cell attachment matrix is prone to geometry changesduring processing. For example, collagen has a tendency to contract ascells proliferate upon it. When unconstrained throughout the cultureperiod, this results in a change in shape after the onset of culture.Post processing is then needed if a predetermined shape is desired. Forexample, in the culture of living skin, it can be desirable to createtissue with relatively straight edges so that the skin can be laid sideby side on a target sight without the presence of untreated gaps. If theskin construct acquires an edge that is not straight due to a shapechange in the cell attachment matrix during culture, post processing tocreate a straight edge can be achieved by cutting the construct. Cuttinga tissue construct to a specific shape after culture can also bedesirable when there are regions of the cell attachment matrix that areunwanted in the final construct. For example, constraining the cellattachment matrix during culture, to retain its shape, maintain it in afixed location for inoculation or optimal feeding, or for any otherreason will lead to sections of the cell attachment matrix that are incontact with the constraining member. Post culture, the sections of thecell attachment matrix that are not in contact with the constrainingmember can differ from the sections of the cell attachment matrix thatare in contact with the constraining member. Thus, it may be desirableto remove that section prior to further use of the construct.

Advantages of these inventions include the ability to perform processingoperations in a closed container, thereby minimizing the risk ofcontamination and damage that may occur from additional handling of theconstruct. FIG. 11 shows an embodiment of a tissue construct bioreactorconfigured to cut a cell attachment matrix to a desired shape withoutany need to first remove the tissue construct from the tissue constructbioreactor. Cutting die 170 resides within tissue construct bioreactor10. Post tissue construct processing, cutting die 170 can be driven intocell attachment matrix 20 in order to cut the tissue construct to adesired shape. A design objective is to perform the cutting processwithout need to open tissue construct bioreactor 10, thereby minimizingcontamination risk. A force can be applied to the upper tissue constructbioreactor wall 30 to drive cutting die 170 through cell attachmentmatrix 20. If tissue construct bioreactor wall 30 is comprised of aflexible material, care should be taken to ensure that the material isnot damaged during the cutting process. If tissue construct bioreactorwall 30 is comprised of a rigid material, the upper tissue constructbioreactor wall can move relative to the lower tissue constructbioreactor wall in order to drive cutting die 170 through tissueconstruct matrix 20. Motion between the upper and lower sections oftissue construct bioreactor 10 should occur without a breach ofsterility. Those skilled in the art will recognize that there are manymethods of driving cutting die 170 in a manner that maintains sterility.Mechanical design solutions that can be employed to create theappropriate motion include piston type movement and die set mechanisms.For example, the techniques used to obtain motion in a die set can byused when tissue construct bioreactor 10 is not structured to allow anymotion between upper and lower tissue construct bioreactor walls 30. Inthis approach, posts attached to the cutting die would project throughone tissue construct bioreactor wall 30 in a sealed manner. Each postwould be capable of moving perpendicular to the plane of the bioreactorwall and be sealed at the point where it projects through the bioreactorwall to maintain bioreactor sterility. Applying a force to the postswould in turn drive the cutting die through the cell attachment matrix.

Cutting die 170 can reside either constrained or unconstrained withintissue construct bioreactor 10. If unconstrained, care should be takento ensure that cutting die 170 does not damage cell attachment matrix 20at any time prior to cutting. Also, care should be taken during thecutting process to ensure that cutting die 170 does not make a partialcut of the tissue construct, as would occur if cutting die 170 perimeterdid not reside entirely upon cell culture matrix 20 at the onset ofcutting. If constrained, cutting die 170 should be free to move in thedirection normal to the plane in which cell attachment matrix 20resides. Side to side movement once cutting die 170 makes contact withcell attachment matrix 20 may be needed to cut cell attachment matrix 20depending on factors such as the strength of cell attachment matrix 20and the sharpness of cutting die 170.

FIG. 12 shows a cutaway through the top view of an embodiment in whichcutting die 170 is configured to cut cell attachment matrix 20 intomultiple sections. Care should be taken to ensure that cell seeding andcell proliferation are not hindered by cutting die 170.

FIG. 13A and FIG. 13B show one example of an embodiment of a tissueconstruct bioreactor comprising two compartments and a cutting dieuseful for separating the desired portion of the cell attachment matrixfrom the undesired portion. In this embodiment, cell attachment matrix20 is constrained by frame 200, creating a portion that is undesirablefor use in the intended application. The desired portion of the cellattachment matrix is separated from the undesired portion while thetissue construct bioreactor remains a closed system. Cutting die 170resides in first compartment 240, positioned such that is does notinterfere with cell seeding or cell proliferation. At a predeterminedstage of tissue construct processing, such as post culture, postcyropreservation, or post shipping the construct to a point of use,cutting die 170 is driven thru cell attachment matrix 20 thereby cuttingcell attachment matrix 20 to a desired shape. In this configuration, asbest shown in FIG. 13B, the portion of frame 200 that resides in secondcompartment 250 is designed to form an interference fit with cutting die170, thereby creating a die set. The illustration of FIG. 13A and FIG.13B show a cutting process when tissue construct bioreactor walls 30 arecomprised of a rigid material. However, the cutting process can alsooccur in a closed system when at least one wall of the tissue constructbioreactor is free to move relative to the opposing wall in a mannerthat does not breach sterility, such as that shown in FIG. 3. In thecase where neither wall of the tissue construct bioreactor is free tomove, cutting die 170 can be driven by way of posts projecting throughthe wall. Care should be taken to ensure that contamination is preventedby sealing the posts to the wall, by using orings or other sealmechanisms known to those skilled in the art.

FIG. 14A and FIG. 14B show cross-sectional views of an embodiment inwhich tissue construct bioreactor 10 is configured to allow cellattachment matrix 20 to be removed. In this configuration, bioreactorlid 260 is hinged to lower bioreactor body 270. Moveable latch 290secures bioreactor lid 260 and bioreactor body 270 together. Lid seal280 prevents contaminants from entering tissue construct bioreactor 10.When moveable latch 290 is repositioned, as shown in FIG. 14B,bioreactor lid 260 can open to allow cell attachment matrix 20 to beremoved. In the case where cell attachment matrix 20 resides in a frame,and has not been die cut, the entire frame can be removed to easehandling.

Cell attachment matrix 20 should be structured for removal according tothe needs of a particular application. Thus, cell attachment matrix 20may be captured entirely about its perimeter, partially captured, orreside in a completely un-captured state. For example, it may bedesirable to capture cell attachment matrix 20 in a frame so that it canbe handled in a manner that does not require direct physical contactwith cell attachment matrix 20. In this manner, physically moving cellattachment matrix 20 can be achieved by making contact with frame 200,and not cell attachment matrix 20. There are also applications in whichit is preferred that cell attachment matrix 20 is completely detached,and those in which it should be partially detached. For example, whenliving skin is to be physically positioned on a patient, any mechanismthat had previously held it in position must first be removed. Doing sowhile it resides within the tissue construct bioreactor, such as by diecutting it, is advantageous in terms of minimizing contamination riskand potential tissue construct damage. If the living skin has a specificside that should face the patient when positioned, a partial cut can behelpful. In this method, the majority of the cell attachment matrixcomprised of living skin would be pre-cut while residing in the closedtissue construct bioreactor. For example, three sides of a square orrectangular cell attachment matrix could be pre-cut while residing inthe tissue construct bioreactor. Subsequently, the living skin would beremoved from the tissue construct bioreactor by handling the frame. Theframe would be designed in a non-symmetric manner, with text,color-coding, or some other features that clearly indicates theorientation of the living skin. The frame and living skin would bepositioned upon the patient in the correct orientation, and the livingskin would be fully removed from the frame by use of an instrument suchas a scalpel.

FIG. 15A and FIG. 15B show the inoculation and feeding stages of anembodiment of a tissue construct bioreactor in which cell attachmentmatrix 20 is held in a desired position by frame 200. Frame 200 hasvertical walls 310, which combined with cell attachment matrix 20 act toform a first compartment 240. In this embodiment, frame 200 isconfigured for gravitational seeding in which all of the inoculumresides directly above cell attachment matrix 20. Vertical side walls310 of frame 200 are designed to retain inoculum 220 in a manner thatminimizes cell seeding onto the walls of frame 200 while positioninginoculum 220 directly above cell attachment matrix 20. Thisconfiguration achieves an objective of minimizing cell deposit onsurfaces other than cell attachment matrix 20. Vertical walls 310 are ofa height such that a predetermined volume of inoculum will be retainedin first compartment 240.

As shown in FIG. 15A, inoculum 220 enters first compartment 240 by wayof access port 40. Cells settle out of inoculum 220 onto cell attachmentmatrix 20. Frame 200 should be designed to capture cell attachmentmatrix 20 in a manner that retards the passage of inoculum 220 throughthe interface between frame 200 and cell attachment matrix 20 and intosecond compartment 250. The amount of inoculum 220 that can pass throughthis interface and still result in an acceptable seeding pattern isdependent upon several factors including cell density, inoculum volume,seeding rate, perimeter length, cell attachment matrix 20 surface area,and cell attachment matrix 20 liquid permeability. If no cell passagethrough this interface is desired, the cell support matrices should beso structured. For example, a 70 Shore A durometer elastomeric gasket,residing on both the upper and lower halves of frame 200, was able toprevent liquid passage when the cell attachment matrix was 0.020 thickbovine collagen squeezed to 50% of its original thickness.

In FIG. 15B, cell culture medium 70 has been added to second compartment250. In this case, the volume of culture medium 70 added to secondcompartment 250 is such that is has risen above vertical walls 310 andoverflowed into first compartment 240. Medium can be isolated in firstcompartment 240 and second compartment 250 by keeping the volume ofmedium below the top of vertical side walls 310. In this manner, onetype of medium could reside in first compartment 240 and another type ofmedium could reside in second compartment 250.

In some configurations, the distance between the cell attachment matrixand the tissue construct bioreactor wall may not be constant. This mayoccur for a variety of reasons. An example would be when the distancebetween the wall and the cell attachment matrix creates a capillaryattraction in the presence of liquid that causes either the wall or thematrix, or both, to be drawn towards each other. Other examples includethe cell attachment matrix not having the mechanical strength to retaina planar position and therefore sagging in unconstrained areas, or thetissue construct bioreactor wall not having the mechanical strength toretain a planar position and sagging in unconstrained areas. If thenon-uniform spacing occurs, inoculation will result in differing seedingdensities across the surface of the cell attachment matrix since thevolume of inoculum residing above the matrix will vary.

In the case where the tissue construct bioreactor wall is flexible andcan be driven towards the cell attachment matrix, such as when externalpressure exceeds internal pressure or when capillary action draws ittowards the cell attachment matrix, it can be constrained such that itresides substantially within a given plane by connecting it to a rigidouter shell. When the tissue construct bioreactor wall is gas permeable,the rigid outer shell should be configured such that the minimal contactwith the gas permeable portion of the tissue construct bioreactor wallneeded to retain the desired degree of planar position is attained. Inthis manner, the maximum gas contact with the gas permeable wall isallowed. In FIG. 16, non-flexible outer housing 330 mates with flexibletissue construct bioreactor wall 30 at predetermined points. Boss 340 offlexible tissue construct bioreactor wall 30 is secured to pocket 350 ofouter housing 330. Care should be taken to ensure that gas compartment140 has an appropriate cross sectional area for the desired application,and that the distance between the tissue construct bioreactor gaspermeable wall 30 and outer housing 330 allows adequate gas exchange.Prototypes using a 100 sq. cm., 0.004 inch thick dimethyl siliconebioreactor wall, provided adequate gas exchange to support 600 billionmurine hybridoma cells when the cross-section was comprised of numerous0.046 inch projections from outer housing 330 that supported a diamondweave mesh comprised of 0.020 inch strands at 16 strands per inch. Theratio of the upper surface of the projections to the surface theyprojected from was about 1:5. Four symmetrically located circular gasaccess openings, 0.32 inches in diameter through the surface of outerhousing 330, covered with 0.2 micron sterile filters, allowed adequatepassive gas exchange for cell culture when the bioreactor resided in a5% CO₂ incubator at 37C. For design estimates, an initial estimate ofthe appropriate cross-sectional area and distance can be made byreferencing a particular cells metabolic activity against the metabolicactivity of murine hybridoma cells, and normalizing for the targetquantity of viable cells using the described dimensional information.

When it is desirable to constrain the flexible gas permeable tissueconstruct bioreactor walls in a substantially planar state, such as whenoriented in the horizontal position while containing liquid or wheninternal pressure exceeds external pressure, boss 340 and pocket 350 canbe eliminated from outer housing 330, as best shown in FIG. 17.

FIG. 18 shows another configuration for maintaining a relativelyparallel geometric relationship and fixed minimum distance between cellattachment matrix 20 and flexible tissue construct bioreactor wall 30.Spacer projections 360 extend from flexible tissue construct bioreactorwall 30 a predetermined distance. Spacer projections 360 act to maintaina fixed minimum distance between flexible tissue construct bioreactorwall 30 and cell attachment matrix 20. To avoid the inhibition of celldeposit and growth, the geometry of spacer projection 360 should be suchthat minimal contact with cell attachment matrix 20 is achieved whilemaintaining the desired degree of parallelism. In this configuration,cutting die 170 is unobstructed by spacer projections 360.

FIG. 19 shows a configuration in which grid 370 is used to maintain arelatively parallel relationship and fixed minimum distance between cellattachment matrix 20 and flexible tissue construct bioreactor wall 30.The geometry of grid 370 should be such that it makes minimal contactwith cell attachment matrix 20 while maintaining the desired degree ofparallelism. Grid 370 can be any type of biocompatible material, and maybe a separate component or part of another component, such as frame 200.Grid 370 should be configured so that exposed sections of cellattachment matrix 20 can be removed when the tissue construct is readyfor further processing or use in an application. Because contact withgrid 370 could affect cell seeding and proliferation, those sections ofcell attachment matrix 20 immediately adjacent to grid 370 should onlybe used after demonstrating that those specific sections are suitablefor use in the intended application.

While the configurations described in FIG. 18 and FIG. 19 allow aminimum distance to be maintained between cell attachment matrix 20 andflexible tissue construct bioreactor wall 30, these configurations maybe operated in excess of the minimum distance by adding fluid volume tothe tissue construct bioreactor, thereby driving flexible tissueconstruct bioreactor wall 30 away from cell attachment matrix 20. FIG.20 shows tissue construct bioreactor 10 in this condition. Note that thespacer projections 360 do not make contact with cell attachment matrix20.

FIG. 21A and FIG. 21B show a method of inoculating one side of a cellattachment matrix residing within a compartmentalized tissue constructbioreactor. In FIG. 21A, second compartment 250 of compartmentalizedtissue construct bioreactor 230 is filled with a predetermined volume ofliquid, such that cell attachment matrix 20 resides in a generallyplanar state when compartmentalized tissue construct bioreactor 230 isoriented in a horizontal position. In FIG. 21B, first compartment 240 isthen filled with inoculum 220. Inoculum 220 can seed onto cellattachment matrix 20 by gravity, while compartmentalized tissueconstruct bioreactor 230 is oriented in a horizontal position, or bycentrifugation as previously described.

FIG. 21A and FIG. 21B, combined with FIG. 22A and FIG. 22B, show amethod of inoculating two sides of a cell attachment matrix residingwithin a compartmentalized tissue construct bioreactor. As shown in FIG.21A, second compartment 250 of compartmentalized tissue constructbioreactor 230 is filled with a predetermined volume of liquid, suchthat cell attachment matrix 20 resides in a generally planar state whencompartmentalized tissue construct bioreactor 230 is oriented in ahorizontal position. As shown in FIG. 21B, first compartment 240 is thenfilled with inoculum 220. Cells from inoculum 220 can seed onto cellattachment matrix 20 by gravity, while compartmentalized tissueconstruct bioreactor 230 is oriented in a horizontal position, or bycentrifugation as previously described. As best shown in FIG. 22A,compartmentalized tissue construct bioreactor 230 is oriented in aposition that allows second compartment 250 to be filled with gas 60 ina manner that displaces the liquid that was present during the initialseeding of cell attachment matrix 20. Subsequently, compartmentalizedtissue construct bioreactor 230 is oriented in a horizontal position andthe liquid of first compartment 240 acts to retain cell attachmentmatrix 20 in a generally planar state. Second compartment 250 is thenfilled with inoculum 220 as shown in FIG. 22B. Inoculum residing insecond compartment 250 can seed onto cell attachment matrix 20 bygravity, while compartmentalized tissue construct bioreactor 230 isoriented in a horizontal position, or by centrifugation as previouslydescribed.

Seeding a cell attachment matrix within a non-compartmentalized tissueconstruct bioreactor, when the density of the cell attachment matrixexceeds that of the cell culture medium, can be achieved by the use ofgravity. As shown in FIG. 23, inoculum 220 is placed in tissue constructbioreactor 10. Cell attachment matrix 20 settles by gravity to theinterior surface of lower tissue construct bioreactor wall 30. Cellssettle out of inoculation by gravity onto the exposed face of cellattachment matrix 20 as shown in FIG. 23A. In the case where it isdesirable to seed two sides of cell attachment matrix 20, inoculum isremoved post seeding of the first side of cell attachment matrix 20 byany of the methods described previously. Thus, tissue constructbioreactor 10 is temporarily in the compressed position of FIG. 23B.Subsequently, tissue construct bioreactor 10 is turned upside down postcell attachment, and a new inoculum 220 is placed into tissue constructbioreactor 10 such that is it exposed to the second face of cellattachment matrix 20 as shown in FIG. 23C. To prevent cell attachmentmatrix 20 from getting out of plane, tissue construct bioreactor walls30 can be flexible, and tissue construct bioreactor 10 can be squeezedsuch that tissue construct bioreactor walls 30 constrain cell attachmentmatrix 20 in a planar state while tissue construct bioreactor 10 isturned over. Subsequently, cell attachment matrix 20 is retained bygravity in proximity to the interior surface of lower tissue constructbioreactor wall 30. Cells settle out of inoculation by gravity onto theexposed face of cell attachment matrix 20.

FIG. 24 shows and embodiment for seeding two sides of a cell attachmentmatrix when it is desirable to minimize cell attachment to surfacesother than that of the cell attachment matrix. As shown in FIG. 24A,frame 200 is configured with vertical walls 310 that retain inoculum 220directly above cell attachment matrix 20 in first compartment 240. Theoutlet of access port 40 is configured such that it resides above aportion of cell attachment matrix 20 allowing inoculum 220 to bedispensed to the interior of the perimeter of frame 200, and thus abovecell attachment matrix 20. If a needle is used to dispense inoculum,access port 40 may reside in a position other that directly over aportion of cell attachment matrix 20 so long as the tip of the needleresides over a portion of cell attachment matrix 20 during inoculum 220delivery. Post seeding, tissue construct bioreactor 10 is turned upsidedown and new inoculum 220 is placed into tissue second compartment 250through a second access port 40, configured as previously described,such that when inoculum 220 is delivered it resides over the exposedsurface of cell attachment matrix 20. As shown in FIG. 24B, cell culturemedium 70 is delivered by way of a first access port 40 such that itresides at a level that covers the lower face of cell attachment matrix20, but does not commingle with inoculum 220. If gas becomes trapped atthe lower surface of cell attachment matrix 20, it can be removed bypenetrating septum 80 of the lower access port 40 with a needle,slightly tilting tissue construct bioreactor 10 such that gas is movedto a the wall of frame 200, placing the tip of the needle into the gas,and withdrawing it. Legs 190 elevate frame 200 such that cell culturemedium 70 can displace gas from the underside of cell attachment matrix20.

Fluid can be delivered into the tissue construct bioreactors describedherein in a batch or continuous manner, and in an automated or manualmanner. In the case of a compartmentalized tissue construct bioreactor,continuous or batch feeding can occur on one side only, on both sides,or across the tissue construct. In the configuration shown in FIG. 25,access needles 380 enter compartmentalized tissue construct bioreactor230 by penetrating septum 80 of access ports 40. Compartmentalizedtissue construct bioreactor 230 is oriented in a vertical position toallow gas to exit. Fluid can be delivered to each compartment from anindependent source, or multiple tissue construct bioreactors can receivefluid from a common source depending on the objectives of a givenapplication.

FIG. 26 shows an embodiment of an invention for minimizing contaminationin applications in which a needle penetrates a septum to access thetissue construct bioreactor. FIG. 25 shows access needles 380 engaged intissue construct bioreactor 230. FIG. 26A shows an enlarged view of oneaccess needle 380 prior to penetration of tissue construct bioreactor230. Access needle protection septum 390 protects access needle 380 fromcontamination. FIG. 26B shows access needle 380 fully engaged in theseptum 80 of access port 40. Access needle protection septum 390 hasrecoiled as a result of contact with the face of septum 80 and hasaltered its position relative to the tip of needle 380. FIG. 26C showsaccess needle 380 after removal from access port 40. Access needleprotection septum 390 has returned to its original position thusprotecting access needle 380 from contamination. Access needleprotection septum 390 can be bellows shaped, spring loaded, or adaptedby any other method that provides a force that drives access needleprotection septum 390 into a position such that its face extends beyondthe tip of needle 380 when needle 380 is not engaged in access port 40.Importantly, the force driving access needle protection septum 390 issuch that the face of access needle protection septum 390 makes contactwith the face of septum 80 during penetration, fluid access, andwithdrawal.

Minimizing the risk of contamination is a very important factor in cellculture as well as in the tissue construct bioreactor designs. More thanone access port can be used to gain access to fluid residing in thetissue construct bioreactors disclosed herein. However, configuring theaccess ports of the tissue construct bioreactor so that airbornecontaminants do not enter the tissue construct bioreactor when adding orremoving fluid can reduce contamination risk. If only one access port isneeded for fluid access, contamination risk can be further reduced. Theaccess port(s) can be configured to seal the tissue construct bioreactorfrom airborne contaminants during fluid handling by designing it tocreate a seal with fluid handing equipment. If at least one of thetissue construct bioreactor walls is comprised of a flexible material,the volume of the tissue construct bioreactor will self adjust as fluidis added and removed. If the tissue construct bioreactor is comprised ofnon-flexible material, fluid can still be added or removed using oneport that is sealed to liquid handling equipment by configuring thetissue construct bioreactor such that its volume can be altered as bestshown in FIG. 3. In this manner, fluids can be added and removed throughone port without changing the pressure within the tissue constructbioreactor.

In the tissue construct bioreactor configurations of this invention,each access port is preferably configured to provide a sealed interfacewith typical fluid handling equipment, such as a pipette, syringe,syringe needle, perfusion circuit tubing, or perfusion circuitmanifolds. For example, a luer lock or penetrable septum would achievethat purpose. When interfacing with a pipette, the access port should beconfigured to allow the pipette to remain attached to the vacuumpipettor when withdrawn. Also, in order to reduce contamination risk,non-sterile surfaces such as the vacuum pipettor or the technician'shand should not reside directly over the access port when the pipetteinterfaces with the access port.

FIG. 27A, FIG. 27B, FIG. 27C, and FIG. 27D disclose configurations of anembodiment for pipette access in a liquid tight manner that allowsnon-sterile surfaces to reside in areas other than directly above theaccess port. This invention is not limited to tissue constructbioreactors, but has use in all applications in which a pipette is usedto add or remove fluid from a container comprised of flexible material,or a non-flexible vented container, or a container capable of adjustingin internal volume. FIG. 27A shows a cross-sectional view of access port40. Access port 40 is designed with an elastomeric thin walled accessopening 42 capable of expanding in cross-section to create a seal withthe tip of a pipette. Threads 48 mate with cap 51 that covers accessport 40 when access port 40 is not in use, thereby preventing airbornecontaminants from entering access port 40.

FIG. 27B shows pipette tip 43 inserted into thin walled access opening42 of access port 40. The cross-section of thin walled access opening 42has increased relative to that of FIG. 27A in order to accommodatepipette tip 43. The seal at the interface of thin walled access opening42 and pipette tip 43 prevents airborne contaminants from entering thetissue construct bioreactor and allows the volume of the tissueconstruct bioreactor to be increased and decreased as fluid is added andremoved. Thin walled access opening 42 applies a seal force to pipettetip 43. The thin-walled nature of the opening is a design characteristicintended to achieve a seal with less force exerted upon pipette tip 43than the force exerted to retain pipette 44 in a vacuum pipettor. Whenthe force required to break the seal between pipette 44 and access port40 does not exceed the force retaining pipette 44 in a vacuum pipettor,pipette 44 will be retained in the vacuum pipettor when it is withdrawnfrom access port 40.

The force needed to dislodge the pipette from the pipettor can varydepending on the pipette, the pipettor, the amount of wear on the rubberpiece in the pipettor that the pipette fits into, and how far theoperator inserts the pipette into the pipettor. To assess the variancein force, a pipette was inserted into a pipettor with as littlepenetration into the pipettor as needed to attain a seal, and as farinto the pipettor as it could go. Then the amount of force needed todislodge the pipette from the pipettor was measured. When the pipettehad minimal penetration into the pipettor, the force required todislodge a 10 ml pipette (Fisherbrand® 13-678-11E) from a pipettor(Integra Biosciences Pipetteboy acu model) was measured at 0.2 lb. Whenthe same pipette had maximum penetration, the force required to dislodgeit from the pipettor was measured at 4.2 lb. The thickness and materialcharacteristics of the thin walled access opening 42 will affect theforce it applies to pipette 44. For example, tests have demonstratedthat when the material thickness of the thin walled access opening is0.02 inches, and the cross-section is circular with an opening diameterof 0.085 inches, and the material has a durometer of 60 Shore A, amaximally inserted pipette will remain in a vacuum pipettor (IntegraBiosciences Pipetteboy acu model) when the tip is removed from thin-wallaccess opening 42 when the pipette has maximal insertion into thepipettor. When the thin walled access opening 42 became wet,approximately 20% less resistance to pipette removal was encounteredindicating that the ratio of material thickness to the diameter of thinwalled access opening could be about 30% (i.e. 0.02 inches divided by0.085 inches times 120%). Void volume 45 is designed such that it makesminimal contact with pipette tip 43 and allows pipette 44 to be insertedat, or rotated to, various angles. Preferably, the majority of grippingforce applied to pipette tip 43 should occur from thin walled accessopening 42 and not from contact with the walls enclosing void volume 45.Fluid access channel 46 allows unencumbered movement of fluid betweenthe tissue construct bioreactor and pipette 44. In applications withcell suspensions, the volume of fluid access channel 46 can be reducedto minimize the number of cells that reside within it. For example, a0.031 inch diameter, 0.5 inches long, will allow adequate flow whilereducing the void volume. If void volume is not a concern, thecross-sectional area of access channel 46 can exceed that of thin walledaccess opening 42. If pipette tip 43 does enter fluid access channel 46,less gripping force will be exerted if fluid access channel 46 is arigid material with a non-circular cross-sectional area, as contact areawill be reduced.

FIG. 27C shows pipette 44 positioned at an angle such that non-sterilesurfaces do not reside directly above access port 40. Pipette stop 47limits the amount of penetration that pipette 44 can make into accessport 40. Threads 48 mate with cap 51 that covers pipette stop 47 whenaccess port 40 is not in use, thereby preventing airborne contaminantsfrom entering access port 40. The opening of pipette stop 47 should bepreferably dimensioned such that when pipette tip 43 resides within voidvolume 45 during fluid handling, and a seal exists between pipette 44and thin walled access opening 42, pipette 44 is prevented from movingfurther into access port 40. When pipette stop 47 is present, anddimensioned in the preferred manner, the cross-section of fluid accesschannel 46 has no effect on the removal force and thus thecross-sectional geometry can be any shape that allows adequate fluidmovement. Pipette stop 47 shrouds access port 40, which is sealed toflexible bioreactor wall 30. As pipette 44 is positioned in order toprevent non-sterile surfaces from residing directly above access port40, the flexible nature of tissue construct bioreactor wall 30, or anyother container comprised with a flexible wall, allows access port 40 tobe angled without any breach of seal.

FIG. 27D shows another configuration of an embodiment in which pipette44 is positioned at an angle such that non-sterile surfaces do notreside directly above access port 40. In this depiction, access port 40sealed to rigid tissue construct bioreactor wall 30, or any other rigidcontainer wall, by way of flexible tube 49. Those skilled in the artwill recognize rotating seals that allow movement of access port 40without breaching sterility are acceptable. The length of pipette stop47 is preferably such that a technician's fingers, or an instrument suchas tweezers, can grip it and move it to a desired angle. Generally, alength of at least 0.25 inches will achieve this purpose. In the casewhere simplicity is desired, and access port 40 is part of a rigidhousing, the entire tissue construct bioreactor can be angled to ensurethat non-sterile surfaces do not reside directly above access port 40.

Another method of preventing non sterile surfaces from residing directlyabove access port 40 during fluid handling is to configure the openingof pipette stop 47 with a dimension slightly larger than the dimensionof thin walled access opening 42 that is required to create the desiredseal of pipette 44, while still limiting penetration into void volume45, thereby minimizing undesired force from being exerted on pipette tip43 by the walls of void volume 45, as best shown in FIG. 27C. In thismanner, pipette 44 can be angled without having to reorient the devicethat access port 40 is attached to. For example, a dimensional openingof pipette stop 47 that is more than 0.010 inches in diameter largerthan that of thin walled access opening 42 can achieve the result wheninterfacing with a 25 ml VWR pipette (catalogue number 53283-710) thatis about 12 inches long.

Further reduction in contamination risk can be attained if the tissueconstruct bioreactor is capable of functioning as a cryopreservationcontainer. To achieve this purpose in the configurations previouslydescribed, the tissue construct bioreactor should be comprised ofmaterials commonly used in cryopreservation bags, such as polyethylene,polypropylene, poly-n-butylene, polyisobutylene, poly-4-methylpentene-1,chlorosulfonated polyethylene, polystyrene, halogenated polyethylene,polymethyl metacrylate, ethylene vinyl acetate, polyvinyl chloride andcopolymers thereof. Ideally, the tissue construct bioreactor should bedesigned such that uniform heat transfer occurs throughout the tissueconstruct. Thus, the walls of the tissue construct should be uniform inthickness to the maximum extent possible. Internal components, such asdies and mechanisms to hold the cell attachment matrix in a desiredposition, should be located such that they do not impede heat transferat the surface of the tissue construct. When possible, access ports thatcause non-uniformity in the tissue construct bioreactor wall should bepositioned such that they do not reside above or below the surface ofthe cell attachment matrix.

Materials typically used in cryopreservation bags may not be suitablefor the tissue construct bioreactor walls in some applications. Forexample, the gas permeability of those materials is inferior tomaterials not commonly used for cryopreservation bags, such as silicone.If the tissue construct bioreactor is comprised of material that can bedamaged during cryopreservation, the tissue construct bioreactor canstill be adapted to allow cryopreservation of the tissue constructwithout any need to first remove the cell attachment matrix and place itin a cryopreservation bag. FIG. 28 shows an embodiment of a tissueconstruct bioreactor with additional cryopreservation adaptation in thecase where the material selected for tissue construct bioreactor walls30 is not compatible with cryopreservation, or when a redundant seal isdesired. Cryopreservation enclosure 130 incorporates tissue constructbioreactor 10. Cryopreservation enclosure 130 can be any materialcapable of withstanding the cell culture cryopreservation process.Cryopreservation enclosure 130 should form a sealed enclosure thatprevents contaminants from entering, or liquid from leaving, theenclosure. Those skilled in the art will recognize that there are manymethods available that are commonly used to form sealed enclosures. Anyadditional materials selected such as adhesives or o-rings must becompatible with the cryopreservation process and are preferablebiocompatible. Preferably, both cryopreservation enclosure 130 andtissue construct bioreactor walls 30 are designed to facilitate uniformheat transfer.

Preferably, the tissue construct bioreactor, cell attachment matrix, andthe cryopreservation enclosure are first assembled and then sterilized.In the case where cryopreservation enclosure 130 does not allow adequategas transfer for a particular tissue construct culture application, gasaccess to tissue construct bioreactor wall 30 is needed by way of gascompartment 140. Gas compartment 140 should be adapted such that gas canmove in and out of it, either by passive diffusion or by forcedmovement. Preferably, gas exchange takes place without breaching thesterility of gas compartment 140, such as by way of sterile gas filter150 residing in gas compartment access port 145. In this manner, thetissue construct bioreactor is capable of providing adequate gasexchange by way of gas permeable tissue construct bioreactor wall 30.Gas compartment spacer 160 prevents contact between tissue constructbioreactor wall 30 and cryopreservation enclosure 130 when either tissueconstruct bioreactor wall 30 or cryopreservation enclosure 130 arecomprised of flexible materials. Thus, a space between tissue constructbioreactor wall 30 and cryopreservation enclosure 130 is maintained,forming gas compartment 140. Preferably, gas compartment spacer 160 isan open structure that retains tissue construct bioreactor wall 30 andcryopreservation enclosure 130 parallel with each other, therebyfacilitating a uniform volume of gas residing within gas compartment 40.It allows gas to make contact with tissue construct bioreactor wall 30to the maximum extent possible. For example, a 0.019 thick diamond weavemesh with 16 strands per inch (Nalle Plastics, Austin Tex.) can be usedto allow relatively unrestricted diffusion of gas throughout gascompartment 140.

Prior to cryopreservation, both tissue construct bioreactor 10 by way ofaccess port 40, and gas compartment 140 by way of gas compartment accessport 145, can be filled with cryoprotectant in a manner that does notbreach sterility. In this manner, if tissue construct bioreactor wall 30loses the integrity of its liquid tight seal during the cryopreservationprocess, it is ensured that cryoprotectant, and not gas, makes contactwith the tissue construct. If sterile filter 150 is not compatible withcryopreservation, it should be removed and aseptically replaced with asecured plug, such as a luer plug, prior to cryopreservation. If anaseptic technique of removing sterile filter 150 is deemed acontamination risk, sterile filter 150 can be configured with a needleto penetrate a septum. Thus, during removal, the system remains closed.

Care should be taken to assure that all surfaces that reside withincryopreservation enclosure 130 remain sterile. That will ensure thatcontamination of the tissue construct does not occur if tissue constructbioreactor wall 30 loses the integrity of its liquid tight seal duringthe cryopreservation process, and cryopreservation material of gascompartment 140 mixes with cryopreservation material of tissue constructbioreactor 10. Preferably, tissue construct bioreactor wall 30 andcryopreservation enclosure 130 parallel with each other leading to auniform volume of cryoprotectant in contact with the surface of tissueconstruct bioreactor wall 30 and thereby facilitating uniform heattransfer.

Cryopreservation enclosure 130 is useful for all bioreactors and cellculture devices that are comprised of gas permeable material that willnot necessarily maintain integrity during the cryopreservation process.For example, cryopreservation enclosure 130 can integrate a cell culturebag of the types described in U.S. Pat. No. 5,686,304 or the VectraCell™marketed by Bio Vectra (Canada). The need maintain sterility is bestachieved by integrating the cell culture bag into cryopreservationenclosure 130 prior to sterilization. Thus, those skilled in the artwill recognize that the previously described attributes and featuresneeded to allow gas access to the cell culture bag, such as those of gascompartment 140, and maintain sterility of the contents of the bag andcryopreservation enclosure 130 including gas compartment 140 when filledwith cryoprotectant should be present.

Those skilled in the art will appreciate that numerous modifications canbe made thereof without departing from the spirit. Therefore, it is notintended to limit the breadth of the invention to the embodimentsillustrated and described. Rather, the scope of the invention is to bedetermined by the appended claims and their equivalents.

1. A cryopreservation and cell culture device including: acryopreservation enclosure, said cryopreservation enclosure being aliquid tight enclosure including at least one access port, and saidcryopreservation enclosure including a tissue bioreactor, and saidtissue bioreactor being a liquid tight container including at least oneaccess port.
 2. The device of claim 1 wherein said tissue bioreactor iscomprised at least in part of gas permeable material.
 3. The device ofclaim 1 wherein a gas compartment is defined between an inner surface ofthe cryopreservation enclosure and an outer surface of said tissuebioreactor.
 4. The device of claim 3 including a gas compartment accessport.
 5. The device of claim 4 wherein the gas compartment includes agas filter, one side of said gas filter in contact with ambient gas andthe other side of said gas filter in contact with gas residing in saidgas compartment.
 6. The device of claim 3 including a spacer disposedwithin the gas compartment.
 7. The device of claim 6 wherein the spaceris a mesh.
 8. A method of cryopreservation using the device of claim 1,said method including the steps of: adding cells and a cell culturemedium into said tissue bioreactor; and culturing cells; and removingsaid cell culture medium and replacing it with a cryopreservationmaterial; and cryopreserving said cells.
 9. A method of cryopreservationusing the device of claim 1, said method including the steps of: addingcells and a cell culture medium into said tissue bioreactor; andculturing cells; and removing said cell culture medium and replacing itwith a cryopreservation material; and adding a cryopreservation materialto said gas compartment; and cryopreserving said cells.